Profiling condition-specific, genome-wide regulation of mRNA stability in yeast

The steady-state abundance of an mRNA is determined by the balance between transcription and decay. Although regulation of transcription has been well studied both experimentally and computationally, regulation of transcript stability has received little attention. We developed an algorithm, MatrixREDUCE, that discovers the position-specific affinity matrices for unknown RNAbinding factors and infers their condition-specific activities, using only genomic sequence data and steady-state mRNA expression data as input. We identified and computationally characterized the binding sites for six mRNA stability regulators in Saccharomyces cerevisiae, which include two members of the Pumilio-homology domain (Puf) family of RNA-binding proteins, Puf3p and Puf4p. We provide computational and experimental evidence that regulation of mRNA stability by these factors is modulated in response to a variety of environmental stimuli

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Puf1p acts in combination with other yeast Puf proteins to control mRNA stability

The eukaryotic Puf proteins bind 3′ untranslated region (UTR) sequence elements to regulate the stability and translation of their target transcripts, and such regulatory events are critical for cell growth and development. Several global genome analyses have identified hundreds of potential mRNA targets of the Saccharomyces cerevisiae Puf proteins; however, only three mRNA targets for these proteins have been characterized thus far. After direct testing of nearly 40 candidate mRNAs, we established two of these as true mRNA targets of Puf-mediated decay in yeast, HXK1 and TIF1. In a novel finding, multiple Puf proteins, including Puf1p, regulate both of these mRNAs in combination. TIF1 mRNA decay can be stimulated individually by Puf1p and Puf5p, but the combination of both proteins is required for full regulation. This Puf-mediated decay requires the presence of two UGUA binding sites within the TIF1 3′ UTR, with one site regulated by Puf5p and the other by both Puf1p and Puf5p. Alteration of the UGUA site in the tif1 3′ UTR to more closely resemble the Puf3p binding site broadens the specificity to include regulation by Puf3p. The stability of the endogenously transcribed HXK1 mRNA, cellular levels of Hxk1 protein activity, and HXK1 3′ UTR-directed decay are affected by Puf1p and Puf5p as well as Puf4p. Together these results identify the first mRNA targets of Puf1p-mediated decay, describe similar yet distinct combinatorial control of two new target mRNAs by the yeast Puf proteins, and suggest the importance of direct testing to evaluate RNA-regulatory mechanisms

Yeast Puf3 mutants reveal the complexity of Puf-RNA binding and identify a loop required for regulation of mRNA decay

The eukaryotic Puf proteins regulate mRNA translation and degradation by binding the 3′ untranslated regions of target mRNAs. Crystal structure analysis of a human Puf bound to RNA suggested a modular mode of binding, with specific amino acids within each of eight repeat domains contacting a single nucleotide of the target RNA. Here we study the mechanism by which the yeast Puf3p binds and stimulates the degradation of COX17 mRNA. Mutation of the predicted RNA-binding positions of Puf3p to those found in Puf5p demonstrated that a single amino acid change in Puf3p abolished detectable binding to COX17. Since this amino acid position in both Puf3p and Puf5p is predicted to contact an adenine in the respective target RNAs, the amino acid in Puf3p must play a more critical role in promoting COX17 interaction. In contrast, an amino acid change in the third repeat of Puf3p, which interacts with the only divergent nucleotide between the Puf3p and Puf5p targets, had no effect on binding COX17. These results argue that a simple set of rules cannot reliably link specific amino acid positions with target specificity. Each of these amino acid changes in Puf3p enhanced binding to the Puf5p target HO RNA, suggesting a different mode of binding to this target. Finally, we identified an outer surface loop that was dispensable for binding but was required to promote both rapid deadenylation and subsequent decapping of the COX17 mRNA, most likely as a point of protein–protein interactions

Relative expression of microRNAs and microRNA host genes in SH-SY5Y cells in wild type and PUM knockdown states.

(A) Expression of host genes of miRs found in Table 1 that contain canonical PRE motifs in their 3’UTRs was measured using RT-qPCR. (B) MicroRNAs determined to be expressed through sRNA-seq were quantified through RT-qPCR. The control bar represents the expression in WT cells. The remaining bars represent the relative expression of the indicated genes or microRNAs in PUM1/2 KD cells. RT-qPCR data was normalized to TUBB for RNA expression and SNORD38B for miR expression. Statistical significance was determined using paired, two-tailed Student’s T-tests. * = p-value ≤0.05, ** = p-value ≤0.01, *** = p-value ≤0.001, and **** = p-value ≤0.0001.</p

KEGG pathway enrichment analysis.

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Sequence conservation within putative canonical (cPRE) and non-canonical (ncPRE) PREs among mammals.

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